Abstract

Protocol to cryopreserve and isolate nuclei from adipose tissue without dimethyl sulfoxide

Author(s): M.M. Almeida1, L.C.J. Caires1, C.M. Musso1, J.M.S. Campos1, C.M.C. Maranduba1, G.C. Macedo2, J.P.R.F. Mendon�§a3 and R.M.G. Garcia1

Cryopreservation injuries involve nuclear DNA damage. A protocol for cryopreserving and isolating adipocyte nuclei is proposed. Adipose tissue samples were directly analyzed (NoCRYO-0h), or stored at -196°C for 7 days without 10% dimethyl sulfoxide (DMSO) (CRYO-WO-DMSO) or with DMSO (CRYO-W-DMSO). To determine the effect of DMSO on cryopreservation treatment, adipose tissue samples were stored at 4°C for 24 h with 10% DMSO (NoCRYO-W-DMSO-24h) and without (NoCRYO-WO-DMSO-24h). Samples were processed in isolation buffer, and nuclear integrity was measured by flow cytometry. The coefficient of variation, forward scatter, side scatter, and number of nuclei analyzed were evaluated. Pea (Pisum sativum) was used to measure the amount of DNA.

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